Quantum Chemical Modeling of Enantioconvergency in Soluble

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Quantum Chemical Modeling of Enantioconvergency in Soluble Epoxide Hydrolase Maria Elin Sofia Lind, and Fahmi Himo ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b01562 • Publication Date (Web): 26 Oct 2016 Downloaded from http://pubs.acs.org on October 26, 2016

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Quantum Chemical Modeling of Enantioconvergency in Soluble Epoxide Hydrolase

Maria E. S. Lind and Fahmi Himo*

Department of Organic Chemistry Arrhenius Laboratory Stockholm University SE-10691 Stockholm, Sweden

Corresponding author: [email protected]

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Abstract Soluble epoxide hydrolases (sEHs) catalyze the hydrolysis of epoxides to their corresponding vicinal diols. One property of a number of these enzymes is that they can catalyze the hydrolysis of some racemic substrates in an enantioconvergent one-enzyme fashion. Here, we have used the dispersion-corrected B3LYP-D3 density functional theory method to investigate the enantioconvergent conversion of styrene oxide (SO) by sEH from Solanum tuberosum (StEH1). A large cluster model of the active site, consisting of 279 atoms, is designed on the basis of the X-ray crystal structure of StEH1 in complex with the competitive inhibitor valpromide. Different substrate orientations of the two enantiomers of SO are examined and the full reaction mechanisms for epoxide opening at the two carbons are calculated, including both the alkylation and hydrolysis half-reactions. The calculated overall reaction energy profiles show that the rate-determining step is associated with the dissociation of the covalent intermediate, which is the second step of the hydrolysis halfreaction. The calculations reproduce the experimentally-observed regioselectivities for the two enantiomers of the substrate, in that both (S)-SO and (R)-SO are calculated to yield the same (R)-diol product. The obtained energy profiles indicate that the transition states for both the alkylation and hydrolysis half-reactions have to be taken into account in order to understand the stereochemical outcome of the reaction. The transition state structures are analyzed in detail and several factors that contribute to the selectivity control are identified. In addition, the mechanistic scenario in which the active site His300 residue is in the protonated form is also considered and the implications on the energies and enantioselection are discussed. The current calculations demonstrate the applicability of the quantum chemical cluster methodology in reproducing and rationalizing experimental enantioselectivities, lending further support to its usefulness as a tool in asymmetric biocatalysis. The results presented here can be helpful in the rational engineering of sEHs to obtain variants with refined biocatalytic properties.

Keywords: enzymology, enantioselectivity, DFT, quantum chemistry, cluster approach, reaction mechanism, transition state


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I. Introduction Epoxide hydrolases (EHs) catalyze the addition of a water molecule to epoxides, forming the corresponding 1,2-diols.1-4 The majority of EHs are independent of cofactors and display activity towards a range of different epoxide substrates, in many cases with high enantioselectivity.5,6 Since both reactants and products are important building blocks in the preparation of enantiopure compounds, EHs are of potential importance for biocatalytic applications, for example in the synthesis of pharmaceuticals.6-13 Indeed, using protein engineering techniques, the performance of several different EHs has been improved in terms of enantioselectivity and regioselectivity.10,12-17 In this context, an interesting feature of EHs is that some enzymes have been found to operate in an enantioconvergent one-enzyme fashion with a number of racemic substrates.8,10,12,13,15-17 For example, soluble epoxide hydrolase (sEH) from Solanum tuberosum (StEH1) has been demonstrated to show enantioconvergent behavior toward styrene oxide (SO) derivatives.18 Namely, the (S)-enantiomers were preferably attacked at the substituted benzylic position, whereas the (R)-enantiomers were predominately attacked at the terminal less stabilized aliphatic carbon (Scheme 1). Owing to this opposite regioselectivity, the (R)-diol is formed in an enantiomeric excess starting out from the racemic epoxide. Similar results have been reported for StEH1 with other substrates using both the wild-type and engineered variants.19,20,21 Other sEHs have also been engineered to affect epoxide hydrolysis in an enantioconvergent manner.22-24

Scheme 1. Enantioconvergent conversion of rac-SO in StEH1. Measured regioselectivity coefficients from Reference [18] are indicated.


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Scheme 2. Generally suggested reaction mechanism of soluble EHs (residue numbering as in StEH1). sEHs belong to the α/β-hydrolase fold superfamily.1-4 The EH members of this family share a conserved catalytic site that contains an aspartate-histidine-aspartate/glutamate catalytic triad and two tyrosine residues.1-4 sEHs are generally suggested to operate through the reaction mechanism shown in Scheme 2 (with StEH1 numbering). The first step, referred to as the alkylation half-reaction, involves a nucleophilic attack by an aspartate residue (Asp105) at one of the oxirane carbons of the substrate to generate a covalent enzyme-


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substrate intermediate.25-30 The active site tyrosine residues (Tyr154 and Tyr235) form hydrogen bonds to the epoxide oxygen and assist thus in the ring opening.31-35 The subsequent hydrolysis of the alkyl-enzyme intermediate takes place in two steps. First, the alkyl-enzyme is attacked by a water molecule, activated by a histidine residue (His300).28,29,30,36 The negative charge on the resulting tetrahedral intermediate is stabilized by an oxyanion hole,37 consisting of the backbone amide groups of the Gly32-Phe33 and Asp105-Trp106 residues. The tetrahedral intermediate is then dissociated to generate the diol product. The histidine is believed to be involved also in this step, facilitating the decomposition of the tetrahedral intermediate by delivering a proton to the product.38,39,40 The combined two-step hydrolysis is referred to as the hydrolytic half-reaction, and has been shown to be rate-limiting for product formation in many EHs.19,30,41,42 A number of computational studies have previously been carried out using different techniques to investigate various aspects of the sEH reaction mechanism and selectivity.39,40,43-51 In a previous quantum chemical study we have used density functional theory calculations employing an active site model of about 100 atoms (considered quite large at the time) to investigate the full reaction mechanism in human sEH.39,46 The calculations lent general support to the mechanism of Scheme 2 in that it was shown to have feasible energy barriers. In particular, it was demonstrated that the tetrahedral intermediate (Int2 in Scheme 2) is in a shallow energy minimum and its collapse (step 3) constitutes the rate-determining step.39 A matter of debate in the reaction mechanism of sEHs has been the protonation state of the histidine residue (His300 in StEH1) during the alkylation half-reaction. According to the general mechanism of Scheme 2, the histidine has to be neutral prior to the hydrolytic halfreaction in order for it to function as a general base in the activation of the nucleophilic water. However, on the basis of molecular dynamics (MD) simulations on murine sEH, it was suggested that the histidine is positively charged in the alkylation step, and that it could be involved in a proton-shuttle pathway that transfers the proton to the negatively charged alkoxide intermediate.45 Our previous quantum chemical study concluded that the possibility of a positively charged histidine during the alkylation step is unlikely, because the calculations indicated that it would result in the this step being endothermic.46 MD simulations and pKa calculations on StEH1 suggested that His300 is predominately positively charged and involved in hydrogen bonding to Asp105 in the substrate-free


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enzyme.40 However, when the substrate enters, the histidine is suggested to be deprotonated via a water-mediated base abstraction by a glutamate residue (Glu35) in the active site.40 More recently, results from quantum mechanical/molecular mechanical (QM/MM) simulations of the alkylation step of murine sEH suggested that histidine is positively charged.48 A neutral histidine resulted in higher energy barriers for the ring opening and also failed to reproduce the correct regioselectivity.48 The calculations provided furthermore an analysis of the factors governing the selectivity of the first step. Very recently, the mechanism and selectivity of wild-type and variants of StEH1 have also been studied using empirical valence bond (EVB) techniques for both the trans-stilbene oxide (TSO)49 and SO51 substrates. Only the first two steps of the mechanism shown in Scheme 2 were considered, and the simulations suggested that the catalytic triad discussed above should be extended by two more residues, His104 and Glu35, as these were found to contribute significantly to the catalysis. The His104 residue was proposed to be protonated, forming an ion pair with the Glu35 residue, and it was found to be important to balance the negative charge that builds up at the active site.49 The regio- and enantioselectivities were also studied with the EVB simulations and the influence of the various residues on catalysis and selectivity were analyzed.49,51 In particular, the enantioconvergence of the SO substrate was reproduced.51 In this case, the simulations indicated that the second step is selectivitydetermining for the (S)-SO substrate, while for the (R)-SO substrate the first alkylation step is selectivity-determining,51 in contrast to the case of TSO for which the regioselectivity of the reaction was found to be determined at the second step.49 Finally, the full reaction mechanism of human sEH was very recently also studied with a limited active site model using semi-empirical and DFT calculations.50 The mechanistic findings were used to explore the epoxide-hydrolase activity of the Ser105Asp variant of Candida Antarctica lipase B. In the present study, density functional theory (DFT) calculations are employed to investigate the sources of the enantioconvergency in StEH1 with rac-SO. A detailed knowledge about these issues could allow for a more rational protein engineering of this enzyme to optimize the selectivity for different purposes. In order to investigate the origins of the selectivity the full reaction mechanism has to be calculated and the details have to be understood. For this, we have designed a very large cluster model of the active site consisting of 279 atoms. The adopted quantum chemical cluster methodology has been employed successfully to investigate the reaction mechanisms of a large number of diverse enzymes.52-56 One implicit 6 Environment ACS Paragon Plus

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question in this context is whether this kind of active site cluster models can reproduce and rationalize the experimental selectivities, for which high accuracy in relative transition state energies is required. We have in a previous study considered the enantioselective conversion of meso-cyclopentene oxide by the wild-type and variants of limonene epoxide hydrolase using a large active site model and the results were in good agreement with experiments.57 The current case constitutes additional challenges for the cluster approach since the substrate is a racemate, and the relative binding and hydrolysis of both enantiomers have to be considered.

II. Computational Methodology II.A. Technical Details All calculations presented in this study were carried out using the hybrid DFT functional B3LYP,58,59 as implemented in the Gaussian 03 package.60 Geometries were optimized employing the 6-31G(d,p) basis set, and more accurate energies were obtained by singlepoint calculations conducted on the basis of the optimized geometries using the larger 6311+G(2d,2p) basis set. The effects of the protein surrounding were estimated by single-point calculations with the 6-31G(d,p) basis set, using the conductor-like polarizable continuum model (CPCM)61,62 with the UAKS radii and a dielectric constant equal to 4. Analytical frequency calculations were performed at the same level of theory as the geometry optimizations to obtain the zero point energies (ZPE). The final energies reported herein are thus potential energies that include also the correction for dispersion effects, calculated according to the DFT-D3 method.63,64 Entropy is neglected in the calculations, as it is expected to have a rather small effect on the energies of the chemical steps of enzymatic reactions.48,65-68

II.B. Active site model A model of the StEH1 active site has been devised on the basis of the X-ray crystal structure of StEH1 in complex with the competitive inhibitor valpromide and ethylene glycol (PDB 2CJP).69 It contains the Asp105-His300-Asp265 catalytic triad and the two active site tyrosines Tyr154 and Tyr235. A number of amino acids constituting the binding pocket are included, namely Gly32, Phe33, Pro34, Trp106, Leu109, Val130, Ile155, Ile180, Phe189, Leu266, Val267 and Phe301. A crystallographic water molecule is also included in the


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model, together with the backbone amide bond of Phe33-Pro34 that forms a hydrogen bond to it. The amino acids are truncated at the α- or β-carbon, except for the tyrosines, which were modeled as phenols. The truncation points, indicated by asterisks in Figure 1, were kept fixed during the optimizations to maintain a structure that resembles the crystallographic one. Hydrogen atoms were added manually. Following the discussions in the previous studies, His300 is modeled in the neutral form.39,40,46,49,50,51 We have also performed calculations on the mechanism and the enantioselectivity assuming a protonated His300 (called Model-P, see below), in accordance with the proposals of Schiøtt et al.45 and Lonsdale et al.48 In addition to His300, there are two other titratable residues in the model that need to be assigned a protonation state, namely Asp105 and Asp265. Asp105 is the catalytic nucleophile and Asp265 is a part of the His-Asp charge-relay pair. Both residues were modeled in their ionized form, in accordance with previous proposals.39,40,45-49,51 In the crystal structure, an additional water molecule is observed within hydrogen bonding distance to both Tyr154 and Tyr235. This water occupies thus the expected position of the epoxide oxygen of the substrate. Therefore, this position was used for the epoxide oxygen when the substrate was inserted into active site model and the water removed from the model. The active site model consists of 279 atoms, including the SO substrate, and has a total charge of −2 (Figure 1). The current model is thus considerably larger than the ones used in the previous quantum chemical studies of soluble epoxide hydrolase,39,46,50 which is necessary in order to investigate the enantioselectivity. In the calculations, both enantiomers of the styrene oxide substrate were considered. As pointed out previously,20,39,44,45,48,50,51 due to the shape of the active site pocket and the substitution pattern of the substrate, one can envisage two different binding modes for each enantiomer. Namely, the phenyl substituent can point either toward the inside of the binding site, hereafter called “in”, or toward the outside and more open part of the active site, called “out” (see Figure 1). For each of these binding modes, the epoxide can be opened at either of the two carbons. Thus, in order to examine the observed enantioconvergency, eight different potential energy surfaces have to be calculated in total, four for each enantiomer of the substrate. For (S)-SO, the energy difference between the two orientations in the enzyme-substrate complexes, called React-S-in and React-S-out, is 0.6 kcal/mol in favor of the latter, while for (R)-SO the energy difference is 2.2 kcal/mol favoring React-R-in over React-R-out. These small energy differences confirm that both binding modes have to be considered in order to investigate the selectivity of the enzyme. 8 Environment ACS Paragon Plus

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Figure 1. Optimized structures of the enzyme-substrate complexes in the active site model with the phenyl substituent of (S)-SO pointing toward the interior of the active site (React-Sin) and toward the exterior (React-S-out). Atoms indicated by asterisks were kept fixed during the geometry optimizations. Note that only polar hydrogens are shown in the figure.

III. Results and Discussion III.A. Catalytic Cycle Using the active site model described above, we have optimized the structures of the transition states (TSs) and intermediates along the reaction pathway outlined in Scheme 2 for the two enantiomers considering both binding modes and attacks at the two carbon centers. The resulting energy profiles for the different possibilities are overall quite similar, as shown in Figure 2. In the discussion here, the steps of the attack at C1 of React-S-in will be used as a representative case (optimized structures of the stationary points are shown in Figure 3), while a detailed comparison between the different binding modes and different opening carbons will be made in the section on enantioconvergency below. In the enzyme-substrate complex (React), the epoxide oxygen is positioned by the two hydrogen bonds to Tyr154 and Tyr235. Two hydrogen bonds are also observed between the side chain of Asp105 and the backbone amide bonds of Asp105-Trp106 and Gly32-Phe33, constituting the oxyanion hole (see Figure 1). The nucleophilic water is kept in position by hydrogen bonds to the Phe33-Pro34 backbone amide and the Nε2 of His300. The Nδ1 of His300 is hydrogen bonding to Asp265, forming the charge relay. Comparing the optimized 9 Environment ACS Paragon Plus

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structure of the enzyme-substrate complex with the crystal structure in complex with valpromide, we note that the two structures are very similar. In particular, the hydrogen bonding patterns observed in the crystal structure are all present in the optimized structure of React. It is also interesting to note that the optimized structure is very similar to the structure obtained in the previous quantum chemical study using a smaller cluster model of the active site.39,46

Figure 2. Calculated energy profiles for the hydrolysis of A) (S)-SO and B) (R)-SO in the “in” and “out” orientations. All energies are relative to the lowest-energy binding mode, which is React-S-out.


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The first step in the reaction mechanism is the nucleophilic attack of Asp105 to form the covalent alkyl-enzyme intermediate (React→Int1 in Scheme 2). The energy of the TS for attack at C1 of (S)-SO in the “in”-orientation (TS1-S-in-C1) is calculated to be only 2.7 kcal/mol higher than the corresponding reactant (React-S-in), and 3.3 kcal/mol higher than the binding mode with the lowest energy (React-S-out), see Figure 2. The resulting alkylenzyme intermediate (Int1-S-in-C1) is 17.1 kcal/mol lower in energy than the enzymesubstrate complex. In the covalent intermediate, a proton is fully transferred from Tyr235 to the developing oxyanion. Next step is the attack of the water molecule on the ester bond of the covalent intermediate to generate the tetrahedral intermediate (Int1→Int2 in Scheme 2). The barrier for this is calculated to be 19.3 kcal/mol. At the TS (TS2-S-in-C1), the water is activated by a proton transfer to the neutral His300 residue (see Figure 3), which in turn is stabilized by the Asp265 residue. The oxyanion hole stabilizes the developing charge at the alkoxide, as demonstrated by the shortening of the hydrogen bonding distances. The resulting tetrahedral intermediate (Int2-S-in-C1) is only 2.6 kcal/mol lower than the TS. The last step is the dissociation of the tetrahedral intermediate to form the diol product (Int2→Prod in Scheme 2). The energy barrier for this step is calculated to be 4.8 kcal/mol relative to Int2-S-in-C1, which is 21.5 kcal/mol relative to the alkyl-enzyme intermediate Int1-S-in-C1. Here, the C-O bond dissociation is assisted by a proton transfer from the His300 to the oxygen of the forming diol (Figure 3). Downhill from the TS, the resulting enzyme-product complex (Prod-S-in-C1) is calculated to have an energy of −17.3 kcal/mol relative to the enzyme-substrate complex. In the enzyme-product structure, Asp105 is in the protonated form, while Tyr235 is deprotonated. To close the catalytic cycle (Prod→React), the diol product has to be released and the active site regenerated, including the restoration of the protonation states and the binding of a new water molecule and a new substrate. These steps are difficult to study accurately with the quantum chemical methodology adopted here. However, an estimate of the overall energetics of these steps can be made by considering the free energy of the overall reaction between the free epoxide substrate and water, which is calculated to be exergonic by 8.0 kcal/mol.70 This means that the overall regeneration process (Prod→React) can be estimated to be endothermic, in the case of React-S-in by 17.3−8.0 = 9.3 kcal/mol. However, it should be


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stressed that this estimation could be associated with large inaccuracies and should be treated with caution.

Figure 3. Optimized stationary points for the hydrolysis of (S)-SO in the “in” orientation at the C1 position. For clarity, only a small part of the model is shown in the figure. For full model, see Figure 1.

The overall reaction energy profiles displayed in Figure 2 show that the rate-determining step is the dissociation of the covalent intermediate (TS3). For the (S)-SO substrate the overall barrier (Int1→TS3) is between 18−25 kcal/mol, depending on binding mode and attacked carbon, and for the (R)-SO substrate it is between 21−26 kcal/mol. These values are overestimated compared to the experimental barriers of c:a 16-17 kcal/mol as determined from the measured rate constants of 3-10 s-1 at 30 °C.19,21,51,71 It is interesting to note that the energy profiles are quite similar to the ones obtained in the previous quantum chemical study using the smaller active site model.39 Apart from the size of the model, one difference in the computational protocol of the current study is the inclusion of the dispersion effect. It is therefore interesting to analyze the effect of this correction. In the SI, the energy graph for React-S-in-C1 is given with and without inclusion of dispersion.


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It is seen that the dispersion lowers the barrier for the alkylation step by ca 3 kcal/mol, and results in a more stable alkyl-enzyme intermediate by ca 5 kcal/mol. The rate-limiting barrier, on the other hand, increases by ca 2 kcal/mol due to the dispersion.

The obtained energetics of the first step are worth additional comments here. The alkylation step is calculated to have a barrier ranging between 3-10 kcal/mol, depending on the enantiomer, substrate orientation and attacked carbon. Experimentally, the rate constant for the alkylation step has been determined to 210 s-1 for (S)-SO51 and >16 s-1 for (R)-SO,19 which can be converted to barriers of c:a 14-16 kcal/mol. The calculated barriers are clearly underestimated compared to the experimental results. However, considering the analysis above regarding the energy associated with the regeneration of the active site for a new catalytic cycle (Prod→React), it can be argued that this endothermicity should be added to the alkylation barrier of the next cycle, in which case it would become closer to the experimental value. It is also possible that the employed computational protocol, in particular the B3LYP functional, underestimates the barrier. Lonsdale et al have shown that the correlated ab initio SCS-MP2 method yields higher barriers than B3LYP, in better agreement with the experimental values.48

A similar analysis can be done for the exothermicity of the alkylation step, which is calculated to be 13-19 kcal/mol (Figure 2). This stabilization of the alkyl-enzyme intermediate is overestimated. Pre-steady-state kinetics experiments of StEH1-catalyzed hydrolysis of (S)-SO indicate that the alkyl-enzyme intermediate is only about 1 kcal/mol lower than the enzyme-substrate complex.51 Again, however, taking into account the energy cost associated with the regeneration of the active site, the exothermicity will be reduced, bringing it closer to the experimental estimate. It should be pointed out that the large exothermicity of the alkylation step seems to be a consistent feature regardless of the size of the quantum chemical model. Both the minimal model employed by Lau et al44 and our previous quantum chemical model39 yielded similar values. The abovementioned EVB simulations gave somewhat conflicting results. For the TSO substrate, the alkylation step was calculated to be almost thermoneutral,49 while for SO it was found to be very exothermic.51 In the first EVB study, it was suggested that the reason for the large exothermicity in the cluster model calculations could be the omission of the positively


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charged His104 in the model.49 To examine the influence of this residue on the energy of the first step, we have extended the model to include the His104 residue, along with its ion-pair partner Glu35 and also the backbone amide of His300-Phe301 to which it forms a hydrogen bond. Very interestingly, the calculated energy turned out to be almost identical to the model without these residues, showing that they have almost no influence on the stability of the alkylation intermediate. The optimized structures and corresponding energies for this model are given in the SI. A factor that potentially could have more influence on the energies of the first step is the protonation state of the His300 residue. As discussed in the Introduction, previous computational studies have suggested that this residue could be in the protonated form prior to the substrate binding and/or during the first step.40,45,48 However, in order for it to act as a general base, it has to lose its proton before the second step. The possibility of a protonated His300 has been deemed unlikely on the basis of the previous quantum chemical study46 and also pKa estimations.40,49 In order to examine the influence of the protonation state of His300 on the energies, we designed a slightly modified model with this residue in the protonated form (called Model-P, see Figure 4, details described in SI) and recalculated the potential energy profile for a representative case, namely that for the attack at C1 of (S)-SO in the “in”-orientation, i.e. starting from React-P-S-in (Figure 5). For the other cases, only the TS1 and TS3 structures were calculated in order to investigate the enantioselectivity (see below).


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Figure 4. Optimized structure of the enzyme-substrate complex in Model-P with the phenyl of the (S)-SO substrate positioned toward the interior of the active site (React-P-S-in).

The overall optimized structure of the enzyme-substrate complex in Model-P is very similar to the corresponding structure in the model with the neutral histidine. Because His300 is in the protonated form, the hydrogen-bonding network is slightly different compared to the neutral case. For example, there is an additional hydrogen bond between the water molecule and Asp105. An interesting observation in this context is that the Nε2 proton of His300 forms a hydrogen bond to the water molecule and not to the Asp105 nucleophile, as suggested by MD simulations.45,48 In our calculations, the hydrogen bond between His300 and the water was formed even when the geometry optimizations were started from structures in which this histidine was hydrogen-bonding to the aspartate. In fact, a hydrogen bond between His300 and Asp105 could only be found when the water was completely removed from the model. Similar observations were made in the previous quantum chemical calculations using a smaller active site model.46 This result, i.e. no hydrogen bond between the histidine and the nucleophilic aspartate, is consistent with a number of crystal structures for other EHs.72-77 There are, on the other hand, also examples of crystal structures in which the histidine in fact is hydrogen bonding to the nucleophile,31,32,38,78 but in most of these structures no water could be observed in that position in the active site. These observations indicate that the presence of


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the water nucleophile in the active site might influence how the histidine is hydrogen bonding. The geometries of the transition state for the nucleophilic attack (TS1-P-S-in-C1) and the resulting intermediate (Int1-P-S-in-C1) are also similar to those of the model with neutral His300 (see SI for structures). Energetically, we note that, while the alkyl-enzyme intermediate is less stable compared to the case of neutral His300 (−9.6 vs. −17.1 kcal/mol), the transition state energy is still very low and very similar to before (+4.0 kcal/mol relative to the binding mode with the lowest energy, see Figure 5). Although there is no direct hydrogen bond between the positively-charged His300 and the negatively-charged Asp105 in the enzyme-substrate complex, the physical proximity between the two charged residues causes a stabilization of this reactant complex as compared to the alkyl-enzyme intermediate, in which the negative charge is located at the Tyr235 residue. This explains the relative destabilization of the Int1 when His300 is protonated compared to when it is neutral. At Int1-P-S-in-C1 the proton on Tyr235 is fully transferred to the oxyanion of the intermediate and His300 is still positively charged. The histidine has therefore to lose its proton in order for it to act as a general base in the activation of the water nucleophile in the next step. One possibility is that the proton moves through some proton transfer pathway to the deprotonated Tyr235 residue.45 We have therefore optimized the geometry for this intermediate (called Int1’-P-S-in-C1), in which both His300 and Tyr235 are in the neutral forms. The energy is calculated to be 4.7 kcal/mol higher than Int1-P-S-in-C1, i.e. −4.9 kcal/mol relative to the reactant (Figure 5). At Int1’-P-S-in-C1, the orientation of the water molecule is the same as in the model with neutral histidine, i.e. forming hydrogen bonds to the backbone amide of Phe33-Pro34 and to the Nε2 of His300.


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Figure 5. Calculated energy profile for the hydrolysis of (S)-SO at C1 in Model-P, starting from the React-P-S-in orientation. Barriers for TS1 and TS3 for (S)-in-C2, (S)-out-C1 and (S)-out-C2 are also indicated. The energies are given relative to the binding mode with lowest energy, React-P-S-out.

The following steps constituting the hydrolytic half-reaction have very similar geometries compared to their counterparts in neutral His300 model, but the energies are somewhat different. The energies for the transitions states for nucleophilic attack (TS2-P-S-in-C1) and the dissociation of the tetrahedral intermediate (TS3-P-S-in-C1) are 16.4 and 18.5 kcal/mol higher than Int1-P-S-in-C1, respectively, to be compared to 19.3 and 21.5 kcal/mol, respectively, with neutral His300. One very significant difference is the energy of the enzyme-product complex in Model-P (Prod-P-S-in-C1). In the model with neutral His300, Prod is calculated to have a similar energy as Int1, while in Model-P, the energy of Prod-P is calculated to be 7.7 kcal/mol higher than the corresponding Int1-P, i.e. only −1.9 kcal/mol relative to the enzyme-substrate complex. In the case of Model-P, the product release and active site regeneration are therefore estimated to be exothermic, by 6.1 kcal/mol, which means that it will not affect the barrier for TS1 in the following cycle, as was argued to be the case for the model with neutral His300. As seen from the overall energy profile for Model-P (Figure 5), the rate-determining barrier also in this case is the dissociation of the covalent intermediate, which has a calculated overall barrier of 18.5 kcal/mol (Int1-P→TS3-P). The protonation of His300 does not solve the issue of the exothermicity and low barrier of the alkylation step.


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As will be shown below, this protonated model does not reproduce the enantioselectivity of the reaction, and is considered less likely also here, in accordance with the previous studies.39,40,46,49,50,51

III.B. Enantioconvergency The calculated potential energy graphs of the entire reaction mechanism (Figure 2) show that TS1 and TS3 have quite similar energies, and both these transition states have therefore to be considered in order to analyze the sources of enantioselectivity. Table 1 summarizes these barriers for both the (S)-SO and (R)-SO substrates. For the (S)-SO substrate, the path with the lowest energy barriers for generating the (R)diol corresponds to attack at C1 of React-S-in, while the path with the lowest energy barriers for generating the (S)-diol corresponds to attack at C2 of React-S-out. The two other pathways (attacks at C2 of React-S-in and at C1 of React-S-out) have higher barriers and do not contribute. The selectivity-determining barrier in the case of (R)-diol stems from TS3 (+4.4 kcal/mol) while in the case of (S)-diol from TS1 (+4.7 kcal/mol). The calculations indicate thus that for the (S)-SO substrate, the formation of the (R)-product (attack at C1) is favored over the formation of the (S)-product (attack at C2), in agreement with the experimental findings, although underestimated compared to the experimental selectivity of 98:2.18 Similar analysis for the (R)-SO substrate shows that the path with the lowest energy barriers for generating the (R)-diol corresponds to attack at C2 of React-R-in, while the path with the lowest energy barriers for generating the (S)-diol corresponds to attack at C1 of React-R-out. The energy difference between the selectivity-determining barriers in this case is 1.4 kcal/mol, in good agreement with the experimentally-measured selectivity of 93:7.18 The results of the calculations show thus that the enantioconvergency of StEH1 can be reproduced satisfactorily with the employed active site model and computational methodology. It is very interesting to note that if one only considers TS1 as being the selectivity-determining step, the enantioconvergency is also reproduced, in fact with even better agreement with the experimental values. Regarding Model-P with a protonated His300 discussed above, we have calculated the energies of TS1 and TS3 for both enantiomers, in the “in” and “out” orientations and for attacks at both carbons. The results for (S)-SO are shown in Figure 5, while the results for (R)-SO are given in SI. It turns out that this model predicts the wrong outcome for both the 18 Environment ACS Paragon Plus

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(S)-SO and (R)-SO substrates. In the case of the (S)-SO substrate, the lowest selectivitydetermining barrier to yield the (S)-diol (TS1-P-S-in-C2) is lower than the lowest selectivitydetermining barrier to yield the (R)-diol (TS3-P-S-in-C1). The energy difference is 0.8 kcal/mol (8.1 vs 8.9 kcal/mol, see Table S1). For the (R)-SO substrate, the lowest selectivitydetermining barrier to produce the (S)-product (TS1-P-R-out-C1) is 0.4 kcal/mol lower than the lowest selectivity-determining barrier to yield the (R)-product (TS1-P-R-in-C2). If one only considers TS1 as the selectivity-determining step, this model predicts the correct enantiomer of the product for (S)-SO, but not for (R)-SO, i.e. the enantioconvergency is still not reproduced. Therefore, taken together, the calculations presented above on Model-P indicate that the scenario of a protonated His300 is less likely.

Table 1. Calculated barriers (kcal/mol) for the nucleophilic attack (TS1) and the dissociation of tetrahedral intermediate (TS3). The energies are given relative to the lowestenergy binding mode, React-S-out. The selectivity-determining transition states are indicated in bold face. Substrate

Orientation

Relative

Attacked

Resulting

TS1

TS3

binding

carbon

diol

C1

(R)

+3.3

+4.4

C2

(S)

+6.7

+2.0

C1

(R)

+9.6

+10.7

C2

(S)

+4.7

+4.2

C1

(S)

+7.1

+11.6

C2

(R)

+4.4

+5.7

C1

(S)

+6.3

+7.1

C2

(R)

+7.2

+7.6

energy (S)-SO

“in”

“out”

(R)-SO

“in”

“out”

+0.6

0.0

+0.1

+2.3

To pinpoint the origins of the differently induced selectivities for the two enantiomers of the substrate, the geometries of all TSs for all orientations must be analyzed and compared. This is a rather complicated task, as the differences in geometries could be quite subtle. However, careful scrutiny of the geometries of the different alkylation transition states (TS1) could help identifying some of the factors that contribute to the selectivity control.


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It is first important to note that the positions of the Asp105 nucleophile and the two tyrosines (Tyr154 and Tyr235) set major limitations to the position and the possible movement of the substrate at the transition state. The Asp105 nucleophile is anchored in its position by hydrogen bonds to the oxyanion hole, while the two tyrosines are somewhat more flexible. Due to these limitations, it can be seen from the optimized geometries that the oxirane ring in all eight different TS1s occupies approximately the same position relative to the nucleophile and the tyrosines. The phenyl substituent of the substrate points in different directions and is rotated differently depending on the enantiomer, the binding mode and the attacked carbon. Apparently, deviation of the oxirane ring from its position is associated with larger energetic penalty than the adjustment the position of the phenyl substituent in the active site pocket. In general, for an optimal attack at the benzylic C1 position, the phenyl substituent should be co-planar with the C1-C2 bond of the oxirane in order to stabilize the developing positive charge. This is indeed the case in TS1-S-in-C1, which is the alkylation TS with the lowest energy and which yields the (R)-product. In this TS, the active site residues allow the dihedral angle between the oxirane carbons and the phenyl ring to be very close to zero as shown in Figure 6. In addition, we can identify two interactions that help stabilizing this transition state. The first is a π-π interaction of a parallel-displaced character79,80 between the phenyl substituent of the substrate and the imidazole ring of the His300 residue (c:a 3.9 Å between the centers of the rings). The second is a CH-π T-shaped interaction79,80 between the side chain of Phe189 and the phenyl substituent of the substrate (c:a 3.4 Å between the center of the phenyl ring of the substrate and the proton in the H-C of Phe189). Previous computational studies have identified similar interactions to be present in the enzyme-substrate complex or in the alkyl-enzyme intermediate.23,44,45,47,51,69,81,82,83 In the other TS1 structures for attack at the C1 position, one or more of these effects are lacking. That is, either the dihedral angle between the oxirane carbons and the phenyl ring cannot assume the ideal value due to steric clashes with the residues in the immediate surrounding of the phenyl ring, or the phenyl substituent points in the “out” orientation and can therefore not form the π-π and CH-π interactions.


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Figure 6. Optimized structures of TS1 for nucleophilic attack at C1 of the (S)-SO (TS1-Sin-C1) and attack at C2 of the (R)-SO (TS1-R-in-C2). The π-π and CH-π interactions are indicated by arrows. Some groups were removed from the figure for clarity.

Similar analysis can be done for the attack at the terminal C2 position. In this case, the phenyl substituent should ideally be rotated to minimize the steric repulsion to the oxirane ring, including the allylic strain. This means that, in contrast to the attack at the C1 position, the dihedral angle between the oxirane carbons and the phenyl ring should not be zero. Thus, in the TS with lowest energy, TS1-R-in-C2, the active site residues permit the phenyl substituent to rotate such that the dihedral angle between the C2-C1 bond and the phenyl ring is 54° and dihedral angle between the O-C1 bond and the ring is 39° (see Figure 6). In addition, this TS has also a CH-π T-shaped interaction between the side chain of Phe33 and the phenyl substituent of the substrate (c:a 3.1 Å between the center of the phenyl ring of the substrate and the proton of the C-H of Phe33). Another important observation here is that in TS1-R-in-C2, the phenyl substituent is also rotated such that it avoids steric repulsion to the side chain of Leu266. In the other TSs with the “in” orientation, this steric repulsion is present, including the above-mentioned TS1-S-in-C1, which has the overall lowest energy. However, in that case, this repulsion is apparently well compensated by the other factors discussed above. Here, it is interesting to note that the π-π interaction with the His300 residue at TS1 is consistent with the fact that mutation of this residue has been shown to affect the kinetics of


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both the alkylation and hydrolysis steps of the reaction.30 In the hydrolysis step, His300 is of course directly involved, acting as both a general base in TS2 and general acid in TS3. To understand the energy differences between the various transition states for the dissociation of the tetrahedral intermediate (TS3), similar analysis as above can be done for their geometries. Namely, they can be analyzed in terms of the rotation of the phenyl substituent relative to the carbon and oxygen atoms of the nascent diol, and also in terms of the existence of possible π-π and CH-π interactions between the phenyl ring and groups in the active site. In addition, a factor that turns out to be useful in explaining some of the energy differences among various TS3s is the rotation around the covalent OAsp105-C bond formed in the first step (O-C1 or O-C2), i.e. whether the substituents are in a staggered or eclipsed conformation.

IV. Conclusions In the present study, the enantioconvergent hydrolysis of styrene oxide by soluble epoxide hydrolase from Solanum tuberosum StEH1 was investigated in detail by means of DFT calculations. A large model of the active site consisting of 279 atoms was designed and the potential energy profiles for the full reaction mechanism were calculated for the two enantiomeric substrates. For each substrate, two different orientations in the active site pocket were examined and attacks at both epoxide carbons were considered for each binding mode, resulting in total in eight potential energy graphs. The obtained energies indicate that the transition states for both the alkylation and hydrolysis half-reactions have to be taken into account in order to understand the stereochemical outcome of the reaction. We found that the calculations reproduce the experimentally observed enantioconvergency, in that the hydrolysis of both (S)-SO and (R)SO yield the same product, the (R)-diol. Detailed analysis of the optimized transition state geometries revealed several factors that contribute to control the selectivity. In addition to the steric and electronic effects inherent to the epoxide-ring opening itself, we could identify a number of interactions between the substrate and various residues of the active site that assist in the transition state discrimination. In the case of the (S)-SO substrate, the active site of the enzyme favors attack at the benzylic C1 position by allowing the phenyl substituent to be co-planar with the C1-C2 bond of the oxirane in order to stabilize the developing positive charge. In the case of the (R)SO substrate, on the other hand, the nucleophilic attack at the C2 position is favored by


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minimizing the steric repulsion between the phenyl substituent and the oxirane ring. Furthermore, the existence of π-π and CH-π interactions between the substrate and the imidazole and phenyl side chains present in the active site provide further stabilization to the two lowest energy pathways that both result in the formation of the (R)-diol product. The insights provided by the current calculations can be helpful in the design of new epoxide hydrolase biocatalysts with improved properties. Here, it should be stressed that it is not straightforward to predict how different mutations of the active site residues would affect the regioselectivity and enantioconvergency, since the mutations can influence the energies of the various TSs differently in the different orientations, and small changes in the active site architecture can cause large changes in the substrate binding and transition state geometries. Finally, the presented results provide further support to the usefulness of the quantum chemical cluster approach in the study of enzymatic reaction mechanisms, including reproducing and rationalizing experimental enantioselectivities.

Supporting Information Results with a model containing the His104 and Glu35 residues. Graph showing the effect of dispersion on PES. Results from the model with a protonated His300. Cartesian coordinates of the optimized structures.

Acknowledgements We acknowledge financial support from the Swedish Research Council, the Göran Gustafsson Foundation and the Knut and Alice Wallenberg Foundation. Computer time was generously granted by the Swedish National Infrastructure for Computing. We thank Profs. Adrian Mulholland, Abraham Mendoza and Nicklas Selander for valuable discussions.

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ACS Catalysis

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ACS Catalysis

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Quantum Chemical Modeling of Enantioconvergency in Soluble

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